LOCUS       sequence-274025-        6183 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  IVT template for the alpha subunit of the mouse TCR #31 that is 
            reactive against MC38.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6183)
  AUTHORS   Gottschalk E, Aksoy BA, Aksoy P, Swiderska-syn M, Mart C, Macpherson
            L, Romeo M, Smith A, Knochelmann H, Paulos C, Timmers C, Wrangle J, 
            Rubinstein MP, Hammerbacher J
  TITLE     Discovering tumor-reactive T-cell receptors through single-cell 
            sequencing of tumor-infiltrating lymphocytes
  JOURNAL   bioRxiv 2021.11.30.470597
REFERENCE   2  (bases 1 to 6183)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2021.11.30.470597"
COMMENT     Sequence Label: pcDNA3.1(+)-MC38-TCRA-31-murderous_crow
FEATURES             Location/Qualifiers
     source          1..6183
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1774..1791)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1780..2004
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2050..2478
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2137..2156)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2347..2368
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2487..2507)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2492..2821
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2672..2807
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2734..2753
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2888..3682
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2942..2961)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3552..3571
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3856..3977
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3893..3912)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3947..3966
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4026..4042)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4026..4042)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4039..4061)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4050..4066
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4074..4080,4081..4098,4099..4104))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4119..4140
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4257..4274)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4428..5016)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4508..4527)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5187..5978,5979..6047))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5810..5829
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6048..6152)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagatgaaaa catatgctcc tacattattc atgtttctat ggctgcagct
      961 ggatgggatg agccaaggtg agcaggtgga gcagcttcct tccatcctga gagtccagga
     1021 gggatccagt gccagcatca actgcactta tgagaacagt gcctccaact acttcccttg
     1081 gtataagcaa gaacctggag agaatcctaa gctcatcatt gacattcgtt caaatatgga
     1141 aagaaagcag acccaaggac tcatcgtttt actggataag aaagccaaac gcttctccct
     1201 gcacatcaca gacacccagc ctggagactc agccatgtac ttctgtgctg caagtgagga
     1261 ggatagcaac tatcagttga tctggggctc tgggaccaag ctaattataa agccagacat
     1321 ccagaaccca gaacctgctg tgtaccagtt aaaagatcct cggtctcagg acagcaccct
     1381 ctgcctgttc accgactttg actcccaaat caatgtgccg aaaaccatgg aatctggaac
     1441 gttcatcact gacaaaactg tgctggacat gaaagctatg gattccaaga gcaatggggc
     1501 cattgcctgg agcaaccaga caagcttcac ctgccaagat atcttcaaag agaccaacgc
     1561 cacctacccc agttcagacg ttccctgtga tgccacgttg actgagaaaa gctttgaaac
     1621 agatatgaac ctaaactttc aaaacctgtc agttatggga ctccgaatcc tcctgctgaa
     1681 agtagccgga tttaacctgc tcatgacgct gaggctgtgg tcctcctaag cggccgctcg
     1741 agtctagagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc
     1801 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     1861 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     1921 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     1981 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg
     2041 ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     2101 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     2161 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     2221 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     2281 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     2341 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     2401 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     2461 aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
     2521 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     2581 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     2641 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     2701 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     2761 tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     2821 aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc
     2881 gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag
     2941 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg
     3001 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa
     3061 tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc
     3121 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc
     3181 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga
     3241 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa
     3301 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct
     3361 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat
     3421 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt
     3481 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta
     3541 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga
     3601 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg
     3661 ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg
     3721 cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     3781 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     3841 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     3901 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     3961 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa
     4021 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     4081 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     4141 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     4201 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     4261 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     4321 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     4381 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     4441 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     4501 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     4561 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     4621 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     4681 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     4741 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     4801 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     4861 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     4921 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     4981 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     5041 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     5101 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     5161 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     5221 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     5281 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     5341 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     5401 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     5461 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     5521 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     5581 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     5641 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     5701 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     5761 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     5821 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     5881 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     5941 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     6001 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     6061 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     6121 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     6181 gtc
//
